The isolation of any significant microorganism from a blood culture is an occurrence that requires careful evaluation by clinicians, and prompt action is usually necessary. The incidence of bacteremia and fungemia had been reported to be 3.4-28/1,000 hospital admissions and was estimated to average 10/1,000 (1%) in the United States (1). The five common isolates from blood cultures were Escherichia coli (E. coli), Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa (2). E. coli is the most important gram-negative bacterium in clinical microbiology laboratories. It is isolated frequently from bacteremic episodes (2, 3, 4, 5, 6), either as a pure culture or as part of a mixed culture. The isolation rate of E. coli can be as high as 40% among the gram-negative bacteria found in bacteremia (6). During the period 1991-1992, in one of the inventors' hospital (National Cheng-Kung University Hospital, Tainan, Taiwan), isolates of E. coli accounted for 21.7% of all bacteria causing bloodstream infections (unpublished data). In another study, E. coli is responsible for 28.4% of all isolates from bacteremic episodes in the pediatric department (7). In view of the high incidence and mortality rate (2), a rapid identification method of E. coli in blood specimens is of clinical importance.
The common practice used in hospitals to identify E. coli is to subculture blood specimens at intervals after an incubation period of 12 hours to 2 weeks (3, 8), and at the time when Gram stain is positive or bacterial growth is apparent (e.g. turbidity, gas production or hemolysis). The subculture and identification steps, however, normally take at least 24 hours. In view of the high incidence and high mortality rate of bacteremia caused by E. coli, in addition to the conventional culture protocols, it is plausible, when the growth of gram-negative bacteria is found, to parallelly perform specific assays to rapidly identify E. coli in blood cultures so that relevant treatment can be started earlier than the conventional identification systems.
Since Kilian and B ulow (9) described the activity of a .beta.-D-glucuronidase (GUD) in restricted species belonging to Escherichia, Shigella, and Salmonella in 1976, this property has been widely used for the detection and identification of E. coli (10, 11, 12, 13, 14) in the food and clinical microbiology laboratories. Most reports incorporated 4-methylumbelliferyl-.beta.-D-glucuronide (MUG) as a fluorogenic substrate of GUD in the media. Some other bacteria (Yersinia, Flavobacterium, staphylococci, and streptococci) are also positive for this enzyme (15, 16, 17), and false-positive reactions are sometimes caused by the animal tissues which contain the enzyme. For this reason, an enzyme-capture assay (ECA) for detection of E. coli in oysters was developed (15). Anti-GUD antibodies coated on the microtiter plate were used to capture the enzyme produced by E. coli present in the food samples, followed by the addition of fluorogenic or chromogenic substrates to demonstrate the GUD activities. Under this condition, cross-reactions are only caused by the members of Enterobacteriaceae.
Although the direct incorporation of a fluorogenic substrate of GUD into a culture media is a convenient and fast way to detect E. coli present in specimens, the practice is impossible for blood cultures, due to the deep red color of blood which masks the fluorescence generated by GUD on the substrate. Enzyme-capture assay seems to be a good choice for solving this problem.
Recently, Husson et al. (18) have described an alkaline phosphatase capture test for the identification of E. coli and Shigella species in blood cultures and urine specimens. Although the specificity (96.8%) is high for detecting E. coli from urine specimens, the sensitivity is relatively low (91%) for blood cultures.
It is still in grave need to develop a rapid method for identifying E. coli in clinical samples with sufficiently high sensitivity and specificity to ensure earlier commencement of antimicrobial treatments.